Spectroscopic Characterization of MIOX

Recombinant MIOX from Mus musculus kidney was recently subjected to a detailed spectroscopic and kinetic characterization. The nature of the iron active site in the presence and absence of substrate wyo-inositol was examined using both EPR and Mfissbauer methodologies [387]. The outcome of this study has not only confirmed that MIOX contains a binuclear non-heme iron center, but also that its diiron cluster is stable in its mixed-valent form and the fully oxidized state. Specifically, the low-temperature X-band EPR spectrum of fully reduced, diferrous MIOX reveals a geff 16 resonance that disappears upon sample exposure to O2, consistent with a conversion of the diferrous center to its mixed-valent form. The 

NATASA MITIC, GERHARD SCHENK AND GRAEME R. HANSON 

Substrate binding also affects the electronic structure of the fully oxidized diferric form of MIOX. Mossbauer spectroscopic data reveal the presence of two antiferromagnetically coupled high-spin Fe ions with a diamagnetic 5,ot = 0 ground state [387]. The addition of /wyo-inositol to the diferric enzyme perturbs the iron active site, but the binding mode is unknown. 

EPR spectroscopy in combination with stopped-flow absorption and rapid freeze-quench techniques has been employed (i) to probe flie catalytically relevant oxidation state(s) of MIOX and (ii) to investigate the reaction between MIOX, substrate, and O2. While most other oxygen-activating binuclear non-heme iron enzymes are catalytically active in their fully reduced form, MIOX exhibits a raflier different behavior. In single-turnover reactions of the diferrous recombinant Mus 

Similarly, the catalytic competence of flic diferric form of recombinant M musculiis kidney MlOX was probed both by EPR and Mossbauer spectroscopy and kinetic studies [391]. Diferric MlOX exhibits very low activity ( 2% of the maximum activity), but is fully active upon treatment with reductants such as L-Cys or ascorbate, which results in the mixed-valent form. In addition, since substrate binding to diferric MlOX was found to be slow and weak, it was concluded that the fully oxidized enzyme was also not a catalytically relevant species. 

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